Zika virus (ZIKV) is an Aedes species, mosquito-transmitted Flavivirus responsible for an ongoing epidemic in over twenty countries and territories in the Caribbean, and in North, Central and South America. In addition to mosquito-borne transmission, ZIKV can likely be transmitted via blood transfusion and by sexual contact with a ZIKV-infected man. Other Flaviviruses, including yellow fever virus and dengue virus (DENV) are known to be transfusion-transmitted and have resulted in severe outcomes. Chikungunya virus (CHIKV), an alphavirus, has a high theoretical risk of transfusion-related transmission. The ZIKV, DENV and CHIKV infection rates are unknown among U.S. blood donors deferred either for travel to an endemic area or for sexual contact with a partner who travelled to an endemic area. Further, the natural history of ZIKV, DENV and CHIKV among deferred blood donors is not well-defined. Enrollment for this study began in mid June 2016 at the NIH Blood Bank, in the NIH Clinical Center Department of Transfusion Medicine. To date, 34 travel-deferred or potential blood donors who traveled to Zika virus endemic areas have been enrolled and tested using highly sensitive and specific RT-PCR-based assays for ZIKV, DENV1-4 and CHIKV (Altona Diagnostics; under FDA emergency use authorization). Enrollees have mean age of 40, 56% are female, 72% are white or Caucasian and 32% had been blood donors >10 years. Donors and potential donors traveled to 23 different countries or territories with endemic Zika virus, including: Argentina, Brazil, Colombia, Costa Rica, Ecuador, El Salvador, Mexico, Panama and Peru; in the Caribbean: the Bahamas, Dominican Republic, Jamaica, Martinique, Puerto Rico and the US Virgin Islands; elsewhere: India, Indonesia, Nigeria, Senegal, Singapore, Tanzania, UAE and Vietnam. We performed internal validation of the ZIKV RT-PCR kit using clinical samples and determined a limit of detection of approximately 100 copies per mL. Thus far, 32 of 34 deferred or potential blood donors were negative for ZIKV, DENV and CHIKV in plasma and urine. One potential donor was positive for Chikungunya RNA in urine (negative in blood) >2 weeks after mild, CHIKV-related symptoms began during a visit to Ecuador. The patient attributed the mild CHIKV symptoms to strenuous volunteer work she was doing for an aid agency in Ecuador, and would not have been reported as an illness during blood donor screening. One was a Zika virus RNA false-positive. She had no significant travel history or other risk factors and subsequent confirmatory testing was negative. We are in the process of preparing a publication of our microarray-based pathogen chip for simultaneous RNA viruses detection, including ZIKV, DENV and CHIKV detection.